Targeting Toll-like receptor-driven systemic inflammation by engineering an innate structural fold into drugs

There is a clinical need for conceptually new treatments that target the excessive activation of inflammatory pathways during systemic infection. Thrombin-derived C-terminal peptides (TCPs) are endogenous anti-infective immunomodulators interfering with CD14-mediated TLR-dependent immune responses. Here we describe the development of a peptide-based compound for systemic use, sHVF18, expressing the evolutionarily conserved innate structural fold of natural TCPs. Using a combination of structure- and in silico-based design, nuclear magnetic resonance spectroscopy, biophysics, mass spectrometry, cellular, and in vivo studies, we here elucidate the structure, CD14 interactions, protease stability, transcriptome profiling, and therapeutic efficacy of sHVF18. The designed peptide displays a conformationally stabilized, protease resistant active innate fold and targets the LPS-binding groove of CD14. In vivo, it shows therapeutic efficacy in experimental models of endotoxin shock in mice and pigs and increases survival in mouse models of systemic polymicrobial infection. The results provide a drug class based on Nature´s own anti-infective principles.

are important for GKY25's antimicrobial activity 1,2 .Furthermore, protonation of H8 at pH 5.5 increases the antibacterial activity of GKY25 against Gram-negative E. coli by membrane disruption 3 .GKY25 binds to LPS, and the LPS-binding hydrophobic pocket of CD14 and the residues responsible for LPS and CD14 interaction have been defined 2 .Studies demonstrate that K14 cross-links to K87 in CD14, and in silico docking studies show that the C-terminal residues of Q17, K18, D21, Q22, and E25 are exposed to the solvent 2 .NMR studies have determined the LPS-bound conformation (PDB:5Z5X [https://www.rcsb.org/structure/5Z5X]) in which the C-terminal a-helix starts at I16. Interactions with LPS are mediated with hydrophobic residues and the positively charged residues H8, R11, K13, and K14 2 .Based on these data, the only suitable amino acid residues remaining available for stapling were G1, L12, Q17, D21, Q22, and E25.
We then performed an in silico analysis of peptide staple positions, whereby a short hydrophobic pentenyl-alanine staple linking either residue i and i+3 or residues i and i+4 was added along the sequence of GKY25.To determine the effect of adding this staple upon binding to CD14, we then calculated the difference in binding energies between the nonstapled and stapled version of the peptide (Supplementary Fig. 1a) (see details in Methods).
We found that the addition of a staple at most positions, particularly on the N-terminal region of the peptide, resulted in poorer binding, as demonstrated by the positive binding energy differences.The reduced affinities could be caused by the staple perturbing interactions between the peptide and CD14.A few staples resulted in improved binding to CD14.For the i-i+3 configuration, these include I16-V19, V19-Q22, I20-F23, and D21-G24, whereas for the i-i+4 configuration, these include V9-K13 and Q17-D21.All the staples that resulted in a more favorable binding to CD14 comprise hydrophobic residues, which could be important for interaction with LPS, except the Q17-D21 staple.Taken together, we, therefore, opted for stapling in the position of Q17 and D21.
We first analyzed the helicity of stapled GKY25 (herein denoted as sGKY25) in comparison to its linear version by using circular dichroism (CD) (Supplementary Fig. 2a).
The spectrum of GKY25 in the presence of LPS was used as a positive control.CD analysis of sGKY25 was compatible with an α-helical structure, and the content of the helicity was comparable to GKY25 when it is bound to LPS.In the case of sGKY25, the α-helical content remained unchanged upon LPS binding.To assess whether stapling increased the proteolytic stability of the peptide, we exposed it to various proteases for different lengths of time, followed by analysis by SDS-PAGE.As shown in Supplementary Fig. 2b, stapling enhanced protease resistance in the presence of HNE for up to 6 h.Protease resistance to trypsin was also increased, with detectable intact sGKY25 after 6 h of digestion.Interestingly, neither of the two peptides showed susceptibility to V8, an enzyme specifically targeting peptide bonds on the carboxyl-terminal side of either aspartate or glutamate 4 .
We next employed LC-MS/MS to understand which regions were released from the digested sGKY25 in comparison to the linear peptide.The results are summarized in Supplementary Fig. 2c and Supplementary Table 1.As expected, the linear non-stapled peptide was extensively fragmented already after 30 min of digestion.Stapling yielded partial protection of sGKY25, particularly in the C-terminal part of the peptide.Notably, stapling of GKY25 increased the cytotoxicity of GKY25 against human monocytes (Supplementary Fig. 2d).Using LPS-stimulated human blood, sGKY25 showed significantly less reduction of TNF-a and IL-ß when compared with the original linear peptide (Supplementary Fig. 2e).sGKY25 was significantly more hemolytic towards red blood cells (RBCs) (Supplementary Fig. 2f).These findings, together with the observation that sGKY25 was only partially protease-resistant, precluded any further development of sGKY25, motivating evaluation of additional variants containing the structurally constrained CD14 interacting region.
Inspection of the in silico data showed that structural locking of G1-F5 was compatible with retained CD14 interaction (Supplementary Fig. 1), and as this would also confer possible protection from aminopeptidases, we, therefore, designed a double stapled GKY25 (denoted 2sGKY25).For this, F5 was replaced by E5, enabling a lactam bridge formation.Moreover, we observed that shorter peptides, corresponding to the original sequence HVFRLKKWIQKVIDQFGE (HVF18) 1 , were formed during the proteolysis of sGKY25.HVF18 is generated by neutrophil elastase in wounds 5 , and its mode of action includes interactions with LPS as well as the LPS-binding groove of CD14 via its critical KKWIQK region 2 .With these background data, we, therefore, decided to analyze stapled HVF18 and related N-terminally truncated stapled versions containing the CD14-interacting region.
peaks in 13 C-HSQC and HN-N amide backbone peaks in the 15 N-SOFAST-HMQC are missing likely due to the oligomerization of the peptide.We, therefore, dissolved the peptide in TFE, which is known to stabilize the secondary structure of peptides 6 . 1 H and TOCSY spectra for sHVF18 in 25% and 50% TFE were overlaid (Supplementary Fig. 6a-b) with similar peak positions and peak shapes for both samples.The 50% TFE sample was selected for further measurements based on the peak width for the stapled peptide linker (Supplementary Fig. 7a-e, respectively).Data were collected for the sHVF18 in 50% TFE.TOCSY, NOESY, and ROESY spectra (Supplementary Fig. 7a-c, respectively) showed welldispersed peaks, where amino acid type can be easily identified in the TOCSY spectra.
NOESY and ROESY spectra show many HN-Ha and HN-HN cross peaks, allowing an easy sequential assignment of the peptide.The 13 C HSQC spectrum (Supplementary Fig. 7d) shows well-dispersed peaks.Sixteen cross peaks corresponding to amide backbone atoms could be detected in the 15 N SOFAST-HMQC spectra (Supplementary Fig. 7e), as well as side-chain cross peaks for W8 and Q15.The 15 N HMQC and 13 C HSQC spectra indicate that sHVF18 presents a well-defined conformational state under these conditions.The presence of multiple HN-HN (I,i+2) and HN-Ha (I, i+2/3) signals indicate a well-defined secondary structure.Assignments were performed, and 97% of the available 1 H resonances could be identified (Supplementary Tables 3 and 4).The DANGLE dihedral angle estimations and chemical shift index (CSI) module in the CCPNMR suite estimated that sHVF18 contains an a-helix consisting of residues 7 to 14, which is also consistent with the NOE pattern.The stapled linker is easily seen in the NOESY spectra due to the aromaticity of the staple (Supplementary Fig. 8) and based on TOCSY spectra, the presence of the staple can be confirmed.Due to the significant peak overlap and complexity of the TOCSY spectra, interresidue NOE cross peaks of neighbor residues were used to identify the sequence of the stapled residues.The sHVF18 3D structure ensemble converged to a backbone RMSD of 0.61 Å. Statistics for sHVF18 are presented in Supplementary Table 5. RMSD for the backbone and for all atoms are shown in Supplementary Table 6.
A comparison of NOESY spectra for sHVF18 in 50% TFE and DMSO-d6, after the assignment of sHVF18 in 50% TFE had been done, indicates that sHVF18 in DMSO-d6 is at least partially folded.This can especially be seen for the HN-HN correlations in the amide region of the NOESY spectra which is indicative of α-helical secondary structures.In addition, cross-peaks are detected between the linker and the amide and aromatic region of the NOESY spectra, further indicating the presence of a folded structure.However, line broadening is severe for sHVF18 in the DMSO-d6 spectra, which is indicative of conformational exchange and prohibiting full assignment.

Supplementary Note 3: Molecular simulations of sHVF18-CD14 interaction.
We performed all-atom MD simulations of sHVF18 bound to CD14 to understand the effect of peptide binding on the dynamics of CD14.Interestingly, our HDX-MS experiments identified regions distal from the N-terminus of CD14 that exhibit deuterium exchange protection upon sHVF18 binding, for example, residues L123-W160.To investigate any potential long-range allosteric effects of peptide binding on the conformation of CD14, we performed simulations of apo CD14 and compared the per-residue root mean square fluctuations (RMSFs) as a measure of flexibility (Supplementary Fig. 14e and f).As expected, a significant decrease in RMSF was observed in residues proximal to the peptide binding site on the N-terminus of CD14 (residues 42-52 and 71-82), consistent with deuterium exchange protection observed in HDX.However, most residues outside of the binding site show overlapping RMSF values, indicating no changes in dynamics during the timescale of our simulations.Longer simulations are likely required to sample any allosteric conformational changes due to peptide binding on CD14.

Supplementary Note 4: Molecular simulations of the sHVF18-lipid A interaction.
Our HDX-MS data demonstrate weakened protection of the putative LPS binding site on the N-terminus of CD14 from deuterium exchange in the presence of sHVF18 and LPS, compared to experiments with LPS alone (Fig. 3c).While our in silico modeling and simulations suggest a competitive binding of the peptide and LPS at the N-terminal rim of CD14 (Fig. 3d and e), another possible mechanism of action is direct neutralization of LPS in solution by sHVF18.Previously, we showed that other similar TCPs, such as GKY25, HVF18, and VFR12, can adsorb and disperse on the surface of lipid A micelles 2 .Lipid A represents the primary bioactive lipid component of LPS; thus, these peptides could encase LPS aggregates in solution and prevent them from interacting with CD14.To investigate if this characteristic is preserved in the stapled peptide, we performed microsecond timescale coarse-grained (CG) MD simulations of sHVF18 with a lipid A aggregate at a 1:2 ratio of peptide to lipid.The simulations included Ca 2+ since divalent cations are crucial to cross-link neighboring phosphate headgroups of lipid A. We found that in all three independent 10 µs simulations, sHVF18 adsorbed onto the surface of the lipid A aggregate and covered a significant portion of the surface, predominantly the exposed lipid tails (Supplementary Fig. 15a).To quantify the degree of surface burial by sHVF18, we measured SASA of the lipid headgroups and lipid tails throughout the simulations and compared it to simulations of a lipid A aggregate without sHVF18 present (Supplementary Fig. 15b).The peptides significantly covered lipid A tails as the SASA was reduced dramatically from 100 nm 2 to less than 20 nm 2 over the course of the simulations.The SASA of the lipid headgroup also decreased from 220 nm 2 to 160 nm 2 .Contact analysis revealed that hydrophobic residues such as F3, L5, W8, and I9 made most contacts with the lipid acyl chains (Supplementary Fig. 15c).While interactions with the polar headgroups of the lipids span almost all residues of the peptide, basic residues, such as H1, R4, K7 and K11, made prominent contacts with the phosphate moieties.Some of these residues form the KKWIQK sequence of the evolutionarily conserved TCP innate fold known to interact with LPS.Apart from direct interactions with lipid A, the sHVF18 peptide also perturbed the cross-links between lipid A headgroups by sequestering the Ca 2+ ions.The number of ions interacting with the phosphate groups of lipid A was notably lower than in simulations without the peptide (Supplementary Fig. 15d).The negatively charged C-terminal E18 residue attracted the Ca 2+ ions away from lipid A (Supplementary Fig. 15e).
We also repeated our CG simulations with E. coli rough Ra LPS containing lipid A with ten additional core sugars present in the headgroup.Similarly, sHVF18 became adsorbed onto the surface of an LPS aggregate, primarily in the lipid A region (Supplementary Fig. 16a), highlighting the importance of the lipid A component of LPS in this interaction.As previously described, the adsorption of peptides onto the LPS aggregate significantly reduced the SASA of the lipid tails (Supplementary Fig. 16b), which was driven by interactions with hydrophobic residues of the peptides (Supplementary Fig. 16c).Similarly, Ca 2+ ions bound to the headgroup were displaced (Supplementary Fig. 16d) due to the interaction of the phosphates with the C-terminal E18 residue of the peptide (Supplementary Fig. 16e).
To further verify our CG MD simulations, we performed atomistic MD simulations whereby one sHVF18 peptide was placed nearby to a small lipid A micelle.As expected, within the first 100 ns, the peptide bound to the surface of the lipid aggregate.Interestingly, the peptide retained most of the secondary structure obtained from NMR, with two α-helices separated by the staple (Supplementary Fig. 18a-c).This suggests that our TFE-derived NMR structure of sHVF18 could represent a physiologically relevant LPS-bound state.Clustering and contact analyses revealed a similar mode of interaction between sHVF18 and lipid A as observed in the CG simulations (Supplementary Fig. 18d and e).The N-terminal basic residues (H1 and R4) of the peptide formed salt bridges with the phosphate groups of lipid A, while the C-terminal acidic residue (E18) formed ionic interactions with the Ca 2+ ions.
Hydrophobic residues along the peptide (F3, L5, I9, and F16) formed hydrophobic interactions with the acyl chains of the lipids.Our atomistic simulations thus corroborate the ability of the stapled peptide to strongly bind to lipid A and encapsulate it, resembling results for linear counterparts as described in our previous study 2 .

GKY25 sGKY25
Sequence 265 266     Y-axis is a difference in uptake (Da), and the x-axis is the peptide number.

Supplementary Fig. 2 |
Biophysical and biochemical analysis of stapled GKY25.a, Representative CD spectra of linear and stapled GKY25 in the presence and absence of LPS.The experiment was performed three times (n=3).The relative a-helical content at 222 nm was calculated from CD spectra.The data are presented as mean ± SEM.Significance was established by an ordinary two-way ANOVA followed by Tukey's multiple comparisons tests using GraphPad Prism software.b, SDS-PAGE of intact and digested peptides with different proteases for different lengths of time.One representative image from three independent experiments is shown (n=3).c, Table and graphical representation summarize all the major peptides obtained after digestion with human neutrophil elastase (HNE) and Pseudomonas elastase (PE) for 30 min and 3 h, using mass spectrometry.Asterisks (*) indicate peptidesa

Supplementary Fig. 6 |bSupplementary Fig. 12 |
NMR spectra of sHVF18 in 25 and 50% TFE.a, 1 H spectra of sHVF18 in 25% (orange) and 50% TFE (green).b, TOCSY spectra (mixing time 80 ms) of sHVF18 in 25% (black) and 50% TFE (red).a Supplementary Fig. 10 | Anti-inflammatory activity of linear and stapled HVF18.a, NF-kB activation and cell viability in THP1-XBlue-CD14 reporter cells stimulated with 100 ng ml −1 of E. coli LPS (LPSEc), 1 µg ml −1 S. aureus LTA (LTASa), 1 µg ml −1 E. coli PGN (PGNEB), 1 µg ml −1 S. aureus PGN (PGNSa), 10 µg ml −1 S. cerevisiae zymosan (ZymSc) in the presence or the absence of 10 µM of linear and stapled HVF18 20 h post-stimulation.Results are presented as mean ± SD of four experiments (n=4).P-values were determined using an ordinary two-way ANOVA followed by Tukey´s multiple comparisons tests using GraphPad Prism software.Binding of linear and stapled HVF18 to CD14. a and c, The differential heatmaps of ligand-free (apo) CD14 with sHVF18 (a) or HVF18 (c) show the uptake perturbations at the 30, 300, 3000, and 9000 s time points.The differential peptide coverage is illustrated by the bars above the heatmap and is color coded to the average deuterium uptake overall observed time points.b and d, Butterfly plot with a central zero showing the residuals between the apo CD14 state and CD14 with sHVF18 (b) or HVF18 (d).The different curve colors indicate deuterium uptake at the different time points (top legend).

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Atomistic simulations of sHVF18 and a lipid A aggregate.A small lipid A aggregate composed of 12 lipid molecules was simulated with a single sHVF18 peptide placed around 2 nm from its surface at the beginning of the simulations.a, Structural comparison of sHVF18 from simulations with lipid A aggregate (left, pink) and sHVF18 from NMR derived in TFE (right, green).A clustering analysis with an RMSD cut-off of 0.4 nm was performed on concatenated MD trajectories and representative structures of the top ten clusters (representing 92% of all simulations) are aligned and compared to the NMR structural ensemble.The staple is shown in grey.b, Average root mean square deviation (RMSD) of the backbone atoms of sHVF18 during the simulations after least square fit to the the NMR ensemble structures.Average values from three independent repeats are shown as a thick line, while the shaded area depicts the standard deviation.c, Average number of residues in the peptide forming an alpha helix during the simulations.The number of residues forming alpha helix in the NMR structure is shown by the dotted red line.d, A representative structure of the peptide-lipid complex.Lipids are shown in van der Waals representation with R L K K W I X K V I X Q F G E Gene set enrichment analysis.Gene set enrichment analysis of 14 gene sets related to sepsis from the "immunologic signature gene sets" database (MSigDB, version 7.3) in LPS 8h compared to controls.

Table 2 .
Summary of all the peptides detected by LC-MS/MS after digestion 261 of linear and stapled HVF18 with human neutrophil elastase (HNE) and Pseudomonas
NF-kB activation and cell viability in THP1-XBlue-CD14 reporter cells stimulated with 100 ng ml −1 E. coli LPS in the presence or absence of increasing concentrations of linear and stapled GKY25, 20 h post-stimulation.Results are presented as means ± SD of four experiments (n=4).Significance was determined by an ordinary two-way ANOVA followed by Tukey's multiple comparisons tests using GraphPad Prism software.e,